The Alphavirus genus in the Togaviridae family contains a number of significant human and animal pathogens that are widely distributed on all continents, excluding the Antarctic regions. Some of the alphaviruses, e.g., the Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses, cause severe human disease. Others, such as Sindbis (SINV) and Semliki Forest (SFV) viruses, are less pathogenic for humans, however, they employ similar replication strategy and are used for studying fundamental issues of the viral RNA synthesis. In the previous grant period, we made a number of important findings toward understanding the SINV genome replication that include i) uncovering the structure and functioning of the RNA promoter elements;ii) demonstration of the critical roles of the Old World alphavirus-specific nsP2 and the New World-specific capsid protein in virus-host cell interactions, in the development of transcriptional shutoff and cytopathic effect in the cells of vertebrate origin, and iii) identification of the viral and cellular components of the SINV-specific protein complexes. Most importantly, our recent studies demonstrated that SINV replication in the cells of vertebrate origin leads to formation of two types of nsP3-containing protein complexes. Based on our data, we hypothesize that the first type of nsP3-specific complexes is initially formed on the cellular membrane and, following endocytosis, remains associated with endosome-like, membrane-containing organelles. These complexes serve as replication complexes (RCs) in virus-specific RNA synthesis. The second type of complexes is associated with the cytoskeleton and has similar viral, but different cellular components, which are normally detected in cellular stress granules (SGs). The latter complexes either function as competitors for SG formation and/or serve in the preferential synthesis of viral proteins. The proposed research plan is focused on two broad Specific Aims: i) to study virus-specific protein complexes formation during SINV replication, and ii) to analyze functioning of cellular and viral proteins in SINV replication. PUBLIC HEALTH RELEVANCE: Alphaviruses comprise a group of widely distributed human and animal pathogens;some of them induce highly debilitating diseases and represent a serious public health threat in the US. The goal of this proposal is to understand the role of the cellular environment in viral infection. Identification of the cellular proteins that are essential for virus growth will lead to development of new antiviral therapeutic strategies.